Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human HeLa cells

F. F. Bukauskas, C. Elfgang, K. Willecke, R. Weingart

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Abstract

A clone of human HeLa cells stably transfected within mouse connexin40 DNA was used to examine gap junctions. Two separate cells were brought into physical contact with each other ('induced cell pair') to allow insertion of gap junction channels and, hence, formation of a gap junction. The intercellular current flow was measured with a dual voltage-clamp method. This approach enabled us to study the electrical properties of gap junction channels (cell pairs with a single channel) and gap junctions (cell pairs with many channels). We found that single channels exhibited multiple conductances, a main state (γ(j)(main state)), several substates (γ(j)(substates)), a residual state (γ(j)(residual state)), and a closed state (γ(j)(closed state)). The γ(j)(main state) was 198 pS, and γ(j)(residual state) was 36 pS (temperature, 36-37°C; pipette solution, potassium aspartate). Both properties were insensitive to transjunctional voltage, V(j). The transitions between the closed state and an open state (i.e., residual state, substate, or main state) were slow (15-45 ms); those between the residual state and a substate or the main state were fast (1-2 ms). Under steady-state conditions, the open channel probability, P(o), decreased in a sigmoidal manner from 1 to 0 (Boltzmann fit: V(j,o) = -44 mV; z = 6). The temperature coefficient, Q10, for γ(j)(main state) and γ(j)(residual state) was 1.2 and 1.3, respectively (p < 0.001; range 15-40°C). This difference suggests interactions between ions and channel structure in case of γ(j)(residual state). In cell pairs with many channels, the gap junction conductance at steady state, g(j), exhibited a bell-shaped dependency from V(j) (Boltzmann fit, negative V(j), V(j,o) = -45 mV, g(j)(min) = 0.24; positive V(j), V(j,o) = 49 mV, g(j)(min) = 0.26; z = 6). We conclude that each channel is controlled by two types of gates, a fast one responsible for V(j) gating and involving transitions between open states (i.e., residual state, substates, main state), and a slow one involving transitions between the closed state and an open state.

Original languageEnglish (US)
Pages (from-to)2289-2298
Number of pages10
JournalBiophysical Journal
Volume68
Issue number6
StatePublished - 1995
Externally publishedYes

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Gap Junctions
HeLa Cells
Temperature
Ion Channels
Aspartic Acid
Clone Cells
DNA

ASJC Scopus subject areas

  • Biophysics

Cite this

Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human HeLa cells. / Bukauskas, F. F.; Elfgang, C.; Willecke, K.; Weingart, R.

In: Biophysical Journal, Vol. 68, No. 6, 1995, p. 2289-2298.

Research output: Contribution to journalArticle

Bukauskas, FF, Elfgang, C, Willecke, K & Weingart, R 1995, 'Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human HeLa cells', Biophysical Journal, vol. 68, no. 6, pp. 2289-2298.
Bukauskas, F. F. ; Elfgang, C. ; Willecke, K. ; Weingart, R. / Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human HeLa cells. In: Biophysical Journal. 1995 ; Vol. 68, No. 6. pp. 2289-2298.
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AU - Weingart, R.

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N2 - A clone of human HeLa cells stably transfected within mouse connexin40 DNA was used to examine gap junctions. Two separate cells were brought into physical contact with each other ('induced cell pair') to allow insertion of gap junction channels and, hence, formation of a gap junction. The intercellular current flow was measured with a dual voltage-clamp method. This approach enabled us to study the electrical properties of gap junction channels (cell pairs with a single channel) and gap junctions (cell pairs with many channels). We found that single channels exhibited multiple conductances, a main state (γ(j)(main state)), several substates (γ(j)(substates)), a residual state (γ(j)(residual state)), and a closed state (γ(j)(closed state)). The γ(j)(main state) was 198 pS, and γ(j)(residual state) was 36 pS (temperature, 36-37°C; pipette solution, potassium aspartate). Both properties were insensitive to transjunctional voltage, V(j). The transitions between the closed state and an open state (i.e., residual state, substate, or main state) were slow (15-45 ms); those between the residual state and a substate or the main state were fast (1-2 ms). Under steady-state conditions, the open channel probability, P(o), decreased in a sigmoidal manner from 1 to 0 (Boltzmann fit: V(j,o) = -44 mV; z = 6). The temperature coefficient, Q10, for γ(j)(main state) and γ(j)(residual state) was 1.2 and 1.3, respectively (p < 0.001; range 15-40°C). This difference suggests interactions between ions and channel structure in case of γ(j)(residual state). In cell pairs with many channels, the gap junction conductance at steady state, g(j), exhibited a bell-shaped dependency from V(j) (Boltzmann fit, negative V(j), V(j,o) = -45 mV, g(j)(min) = 0.24; positive V(j), V(j,o) = 49 mV, g(j)(min) = 0.26; z = 6). We conclude that each channel is controlled by two types of gates, a fast one responsible for V(j) gating and involving transitions between open states (i.e., residual state, substates, main state), and a slow one involving transitions between the closed state and an open state.

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